The Effect of Non-Surgical Periodontal Therapy on Pentraxin-3 Concentration in Gingival Crevicular Fluid of Chronic Periodontitis Patients: A Clinical Trial

Statement of the Problem: Periodontitis is a multifactorial disease caused by periopathogens and its severity is determined by the host immune response. Gingival crevicular fluid (GCF) can be used for non-invasive testing to assess the host response in periodontal treatment. Pentraxins are the classic mediators of inflammation and pentraxin-3 can be used as a marker to assess response to therapy, which was investigated in this study. Purpose: This study aimed to assess the effect of non-surgical periodontal therapy on GCF level of pentraxin-3 in patients with chronic periodontitis. Materials and Method: 25 patients with chronic periodontitis (CP) and 25 periodontally healthy controls were evaluated. Pocket probing depth, clinical attachment loss, plaque index, gingival index, and bleeding on probing were measured in both groups. GCF samples were collected using paper strips to assess the level of pentraxin-3. In the CP group, GCF samples were collected from the highest clinical attachment loss, pocket probing depth, and bone loss at baseline and six weeks after non-surgical therapy. The level of pentraxin-3 in the GCF was quantified by enzyme-linked immunosorbent assay (ELISA). Data were analyzed using SPSS version 23. Results: Pentraxin-3 in GCF of CP patients before treatment (6.72±4.63 ng/mL) was higher than the control group (4.43±2.85 ng/mL). Pentraxin-3 in patients after non-surgical therapy (3.2±2.66 ng/mL) decreased significantly compared to the baseline (p= 0.04) and its level after treatment was not significantly different from the control group (p= 0.14). Conclusion: Pentraxin-3 in GCF of CP patients was higher than healthy controls and decreased in response to non-surgical periodontal therapy. Thus, it can be used as an inflammatory marker for detection of patients at risk of CP. However, further studies with larger samples and longer follow-ups in different populations are required to confirm our findings


Introduction
Periodontitis is a chronic inflammatory disease that is caused by polymicrobial infection [1]. It results from the interaction of periodontal pathogens and host inflammatory responses [2]. Chronic periodontitis (CP) is often diagnosed based on radiographic findings and clinical parameters, including pocket probing depth (PD) and clinical attachment loss (CAL). However, measurement of these parameters does not provide any information about the current status of disease or its future course [3]. Biomarkers are capable of providing additional information on standard clinical indices [4][5].
The progression of periodontal disease is sitespecific and an increase in the count of lymphocytes, neutrophils, and macrophages is seen in primary lesions [6]. The gingival crevicular fluid (GCF) also increases during gingival inflammation [5,7]. Periodontal inflammation is regulated by a combination of chemokines and cytokines [8]. During the primary phases of CP, bacteria invade the connective tissue [9]. In response to bacterial endotoxins, acute phase proteins are produced that activate an inflammatory cascade [10][11].
Inflammatory reactions increase the level of acute phase proteins in GCF [12].
Pentraxins can be categorized into two groups; the first group is short pentraxins, and the second is long pentraxins [13]. The first group includes C-reactive protein and serum amyloid P component. They are acute phase proteins produced in response to inflammatory mediators in the liver [14]. The first long pentraxin is Pentraxin-3 [8], which plays a vital role in the inherent resistance to pathogens, regulation of inflammatory reactions, and removal of apoptotic cells [9]. Pentraxin-3 is produced in response to pro-inflammatory signals such as bacterial constituents (lipopolysaccharides, lipoarabinomannans, and outer membrane proteins), Toll-like receptor engagement, and cytokines (tumor necrosis factor-alpha and Interleukin1β) [15]. It is produced and released by diverse cell types, primarily by cells frequently found in the periodontal tissue, including fibroblasts, neutrophils, dendritic cells, epithelial cells, macrophages, and endothelial cells [4,16].
One of the main challenges in periodontology is finding a reliable high-sensitivity and -specificity molecular marker for early detection of periodontal tissue destruction. In the recent years, a number of cellular mediators and enzymes have been identified in the GCF with predictive value in detection of tissue loss in CP [4,[17][18]. Due to extra-hepatic synthesis of pentraxin-3 in the inflamed tissues (in contrast to C reactive protein), it is believed that the level of pentraxin-3 is an independent indicator of disease activity [19]. In addition, pentraxin-3 can activate the complement system, which highlights its role in regulation of inflammation and innate immune responses [20]. A review study done by Katharia et al. [8] showed that pentraxin-3 might be used as biomarker in the diagnosis of periodontal disease; besides, it might have clinical implication in diag-nosing the "at site" inflammatory status of periodontal disease.
Since there is paucity of research on the effects of non-surgical periodontal therapy (scaling and root planing) on GCF level of pentraxin-3, this study aimed to assess the effect of non-surgical periodontal therapy on GCF level of pentraxin-3 in patients with chronic periodontitis [21][22].

Materials and Method
The study was approved in the Ethics Committee of Full-mouth clinical periodontal examination (except for third molars) was carried out for both the patient and control groups including the assessment of PD and CAL at 6 sites around each tooth using a Williams periodontal probe (Hu-Friedy, Chicago, IL). The plaque index (PI) [24], bleeding index (BI) [25] and gingival index (GI) [26] were recorded. In order to avoid GCF contamination with blood resulting from the probing of inflamed sites, samples were collected on the subsequent day.
Participants then received oral hygiene instructions (the rolling method twice a day). Phase one periodontal therapy was performed for CP patients in two sessions with one-week interval, and included scaling and root planing with hand and ultrasonic instruments. No antibiotic or anti-inflammatory agent was prescribed for the patients. CP patients were recalled six weeks after the second treatment session. GCF was collected again from the same areas and clinical parameters (CAL, PD, PI, BI, and GI) were measured again. All procedures were performed by the same operator (a post-graduate student of periodontology). The patients were recalled two weeks after the second scaling session and their adherence to oral hygiene instructions was evaluated.
Supragingival prophylaxis was also performed for them.

Collection of GCF
Periopaper collection strips (Oraflow Inc., Smithtown, NY, USA) were employed for GCF collection. In CP patients, GCF was collected from the region with the highest CAL, inflammation, and radiographic bone loss.
In healthy controls, GCF samples were collected from non-inflammatory areas with PD<3 mm without BOP.
To prevent saliva contamination, isolation was performed with cotton rolls. A gauze was employed for the removal of supragingival plaque and the teeth were dried with air spray. Strips were inserted in to the crevice until mild resistance was felt and remained there for one minute. Samples with detectable blood contamination were excluded. Then, collected paper strips were placed in micro-centrifuge tubes and stored at -80°C until laboratory analysis. All samples were coded and sent blindly to the laboratory. Samples were collected at baseline before scaling and root planing and also at 6 weeks after the completion of phase I periodontal therapy.

Assay procedure
The level of pentraxin-3 in GCF samples was quantified

Statistical analysis
Data were analyzed using SPSS 23 (SPSS Inc., IL, USA). Kolmogorov-Smirnov test was used for testing the normality of data distribution. The two groups were compared using a Student t-test, and paired t-test was used for comparisons within the groups. The Pearson's correlation test was applied to assess the correlation between the clinical parameters and concentration of pentraxin-3 separately in the control and patient groups before and after the treatment. P ≤0.05 was considered to be statistically significant. The power of the study was determined as 80% and α=0/05.

Discussion
This study aimed to assess the concentration of pentraxin-3 in CP patients before and after non-surgical periodontal therapy compared to the control group. The number of males and females was the same in the CP and control groups. Also, the participants were in the range of 25 to 50 years. It minimized the effect of gender and age on the results. The current findings confirm-    Minimum values were recorded in the healthy group.
In a study by Elmonem et al. [27], the mean concentration of pentraxin-3 in GCF of CP patients was significantly higher than patients with gingivitis and healthy controls. Also, the concentration of pentraxin-3 was significantly correlated with clinical parameters. Fujita et al. [6] showed a strong correlation between the concentration of pentraxin-3 in GCF and site clinical parameters. In current study, only the site PD had a significant correlation with the concentration of pentraxin-3 in the GCF, which may be due to different sample size, inclusion and exclusion criteria, and study design, as Fujita et al. [6] followed a split-mouth design.
To the best of authors' knowledge, only two previous studies performed by Afifi et al. [22] and Mohan et al. [21], evaluated the level of pentraxin-3 in GCF of patients with CP before and after non-surgical periodontal therapy. In the study by Afifi et al. [22], GCF samples were collected at baseline and 8 weeks after treatment completion. They showed significantly higher concentration of pentraxin-3 in CP group compared to the control group at baseline. The concentration of pentraxin-3 in the CP group significantly decreased after treatment compared to baseline, but it was still higher than the control group.
In the present study, the mean concentration of pentraxin-3 after treatment was not significantly different in CP and control groups. Afifi et al. [22] only evaluated female patients and collected samples at 8 weeks while we evaluated both males and females and collected samples at 6 weeks after completion of treatment. Also, patients were recalled after 2 weeks and received supragingival prophylaxis. These factors may explain the difference in the results of the two studies.
In our study, the GI decreased from 68% at baseline to 19.8% after treatment. This reduction was significantly greater than that reported by Afifi et al. [22], since in their study, GI decreased from 66% to 31%. This indicates greater resolution of inflammation in our study and subsequently greater reduction in the concentration of pentraxin-3 in GCF and absence of a significant difference in its concentration between the CP and control groups.
A study by Mohan et al. [21] investigated the effect of nonsurgical periodontal therapy on pentraxin 3 levels in smokers and nonsmokers with CP. In this study, Pentraxin -3 levels at baseline in patients with CP (smokers and non-smokers) were found to be significantly higher than both groups of clinically healthy subjects (smokers and non-smokers). They showed that scaling and root planing improves clinical parameters and significantly reduces Pentraxin-3 levels (p< 0.05) in both chronic periodontitis groups at 2-week follow-up, but these changes were more significant in smokers compared to nonsmokers.
The current study, consistent with Mohan et al. [21], only assessed non-smokers; hence, pentraxin-3 concentration in the CP group was higher than in the pre-treatment control group, while it significantly decreased after the periodontal therapy. Periodontitis is often diagnosed based on radiographic findings and clinical measurements, which is cost-effective but does not provide any information regarding the current status of disease and prediction of its course in the future. Thus, a main challenge in periodontology is to find a reliable molecular marker for periodontal tissue destruction with high sensitivity, specificity, and applicability [28].
Considering all the above, it appears that the concentration of pentraxin-3 in GCF may be used to assess the disease status and response to periodontal therapy.
However, future studies are suggested to be conducted with larger sample sizes and longer follow-up periods in different populations to increase the reliability and generalizability of our findings. Also, the concentration of pentraxin-3 should be evaluated in GCF of CP patients before and after periodontal surgery. Assessment of the level of pentraxin-3 in the saliva of CP patients before and after surgical and non-surgical periodontal therapy can also be an interesting topic for future research in this field.

Conclusion
A higher concentration of pentraxin-3 in GCF of CP patients than in healthy controls may highlight the role of this marker in the pathogenesis of periodontitis.
Therefore, it can be used as an inflammatory marker in periodontal disease and can be employed to detect individuals at risk for CP. Nevertheless, Pentraxin-3 concentrations varied considerably in different groups.
Therefore, to consider Pentraxin-3 as an inflammatory biomarker of periodontitis in GCF, further studies with larger sample sizes and more extended follow-up periods in different populations are required.